In-situ biochip and its preparing process

Embodiment 1

The invention relates to an in-situ biochip, as shown in Figure 1-5. On it by the substrate and an array of detection sites.

The above-mentioned substrate 1, assumes evenly sheet.

The substrate 1 is, as shown in Figure 1, along the X coordinate axis of the RX address line M of RX1, 1X2, RX3 the address   RXM, and along the Y axes of the address lines RY N RY1, RY2, RY3... RYN address X of Y the resistance and is connected with the resistor 2 a resistor array, the above-mentioned M, N is a positive integer, the RX RY address lines of the address line and one end of the are respectively and X resistance and Y resistance 2 is connected, the other ends are respectively the usual X resistance logic circuit 3 and Y resistance logic circuit 4 is connected, for probe positioning synthetic. The resistance array is covered with a layer of SiO₂ film layer 5. SiO₂ film layer with the resistor array on the corresponding X coordinate axes including M of the X address lines X1, X2, X3... XM address line, and along the Y coordinate of the Y address lines N of Y1, Y2, Y3... YN address line is staggered, the above-mentioned M, N is a positive integer. The X address lines and Y address lines is interlocking Department MN MN of a sample solution can contain 6 detection site 7. The electrode in the detection site, as shown in Figure 3, 4 shown, there is at least one of a finger-like strip of the upper electrode 8, and at least one takes the form of finger-like strip located on the lower electrode 9. The electrode 8 and Si0₂₊ film 5 have an adhesive layer in sequence between 10 and Si₃ O₄ mask layer 11, the electrode 9 and SiO₂ film 5 have an adhesive layer between the 10. A plurality of electrode 8 and a plurality of electrode 9 a parallel, spaced interdigitated electrode groups. The detection site of the two adjacent ones of the electrode of the upper layer 8 with the lower layer of the electrode 9 formed between the pool 12, in the detection site to form a plurality of detection cell. The above-mentioned X address line and one end of the Y address lines respectively connected with the electrode 8 and electrode 9 connected with, another end is connected with the usual X addressing logic circuit 13 and Y addressing logic circuit 14 is connected. X addressing logic circuit and Y addressing logic circuit of the detection circuit 15 are connected to form a plurality of sensors, and the above-mentioned resistor array relative to the sensor array. In a sensor detection pond is cured on the electrode of a probe 16, sensor in each sensor in the array of the probe to form the array searches the row , in the probe array probe can be organic compounds, biological molecule probe, probe cells, microorganism at least one of a probe. Thus a detection site array. The above-mentioned X resistance logic circuit 3, Y resistance logic circuit 4, X addressing logic circuit 13, Y addressing logic circuit 14, the detection circuit 15 are integrated in the substrate 1 is.

The invention of the above-mentioned in-situ biochip preparation method is as follows.

1) the above-mentioned substrate 1, made of insulating or semiconductor material and usually even laminated form prepared according to the method, the material used can be single crystal silicon, glass, quartz, and corundum piece.

2) on the substrate of the large scale integrated circuit technology to the manufacture of miniature sensor array.

(1) on the substrate 1 the upper vapor deposition or sputtering resistance material such as nickel-chromium alloy, tungsten or platinu metal along X coordinate axes RX address line M of RX1, RX2, RX2... RXM address line, and along the Y coordinate of the address line RY N of RY1, RY2, RY3... RYN address line, and is respectively connected with the RX address lines and RY address X of Y the resistance and is connected with the resistor 2 a resistor array, as shown in Figure 1. Resistor 2 can also be through a number of mature technologies such as chemical vapor deposition (CVD), molecular beam epitaxial (MBE), metal organic CVD (MOCVD), or other semiconductor process, a doped polysilicon, silicide or of tungsten either tantalum or platinum is made of nitride or nitrogen oxides. RX RY address lines of the address line and the other end of the integrated on the substrate with the usual X resistance logic circuit 3, and Y resistance logic circuit 4 is connected.

(2) the use of chemical vapor-phase deposition method, the thickness of the resistance array is about 5000 the of (angstroms) [...] SiO₂ film layer 5, then in SiO₂ layer, M generating along X coordinate axes of the X address lines X1, X2, X3... XM address line, and along the Y coordinate of the Y address lines N of Y1, Y2, Y3... YN address lines, then in SiO₂ mask material on the film Si₃ O₄ produce a thickness of about a 500 of [...] Si₃ O₄ mask layer 11.

(3) the electrode pattern using photolithography method, then the reactive ion etching method, according to the electrode pattern corrosion removing Si₃ O₄ mask layer 11 is produced in the X address lines and Y address a MN beams is composed of a plurality of mutual parallel to the circular groove 2 the hole of the long strip   m 17 hole Kong Zu composed of, as shown in Figure 5B illustrated.

(4) with an acidic buffer fluid such as HF corrosion depth buffer liquid about 4000     SiO₂ film layer 5, the hole 17 is the bottom surface of the pocket 18, as shown in Figure 5C illustrated.

(5) by electron-beam evaporation, respectively, the bottom surface of the in all atractaspis hole 18 the SiO₂ film 5 surface, and the remaining Si₃ O₄ mask layer 11 surface, produce a thickness of about the 300 [...] adhesive layer 10, and then on the adhesive layer thickness of the material in the generating gold about 2000 the metallized layers of [...] , located respectively obtain Si₃ O₄ mask layer 12 on the electrode formed by the metallization layer 8, and are arranged on the bottom surface 18 of the electrode 9, formed by a plurality of finger-like strip-shaped electrode 8 and electrode 9 a are interdigitated electrode groups. The bottom surface of the hole and/or the surface of the electrode, can be made into a corrugated surface, the textured configuration of the detection site surface area increase, thus can be fixed more probe in order to obtain more high degree of sensitivity. As shown in Figure 3 a series of interdigitated electrode about the 100  m, width is about 100 the  m, each of the electrodes to about 2 the long strip-shaped   m, the electrode spacing about 2 the  m. Such interdigital design can be the very small zone on the substrate in the sample of a large electrode surface area of contact, thereby having higher capacitance "sample" of the detection site electrode lead to the ratio of the parasitic capacitance. The electrode 8 and electrode 9 and is connected with the X address lines and Y address, X address line and the other end of the Y address lines of the integrated on the substrate are 1 X on the addressing logic circuit 13 and Y addressing logic circuit 14 is connected, then the X addressing logic circuit and Y addressing logic circuit and is integrated in the substrate 1 of the detecting circuit 15 is connected, a sensor MN, composition sensor array, Figure 5A-5D is only given by the substrate 1 on a part of a detection site 7. The introduction of modern semiconductor technology, the diameter 3 inches of the processing on the substrate 700 million or more such a detection site.

3) is fixed on the detecting site probe

The probe of the detection sites is based on determining the target getting unknown, can adopt different probe, including oligonucleotide, single or double-stranded DNA or RNA antibody or antigen-antibody complexes, tumor cells can be any other and combined with each other to the target substance of the material. For example, RNA or DNA target material, the probe can be a synthetic oligonucleotide. In a detection site on the there should be a probe, is formed by a plurality of detection sites on the chip array formed by a plurality of probes corresponding to a probe set. This invention adopts the organic compound, biological molecule, cell, at least one microorganism in a combined micro display as a probe, capture at the same time, detection probe interaction with a target substance. The target material comprises a plurality of corresponding group of a target material is detected at the same time.

As shown in Figure 4, the probe of this invention 16 is directly fixed on the detecting site 7 the pool in the 12 inner electrode 8 and electrode 9 is. Probes can also be fixed on the solid support (not shown in Figure) is, in use, the solid support is placed into a detection cell together with probe 12 the inner. Solid support, the substrate can be organic inorganic matter, such as glass, polystyrene, poly-amide, silicon dioxide and silicon nitride. The fixed probe solid support or electrodes need to be pre-functionalized in order to produce with the selected probe is covalently attached to the surface chemistry. For example, glass are used for the fixed support, by the reaction of the epoxy group of the epoxy silane functionalized, with the glass of the epoxy group on the 5 [...] -amino-derivatized oligonucleotide probe reaction, covalent coupling to form a secondary amine, and the probe is coupled to the glass surface. If the 5 aldehyde or carboxylic acid, amino and phosphate derivatives can be respectively connected with the hydrazide, diazotization activation and combination of polystyrene modified with nitrogen. The fixed probe the surface of the electrode also need to be pre-processing coating can be directly combined with the probe on the material, these materials include gold, niobium oxide, iridium oxide, platinum, titanium, tantalum, tungsten and other metal, and these metallic surface made on the probe forms a stable organic of the coupling is connected. Such as a 5 the or [...] 3 the hydrosulphonyl [...] end mark on the synthesis of the DNA probe can form a stable conjugate metal payment.

In the synthesis of the probe can be fixed after the various probe, adopting the micro-pipette or micro-nozzle needle or the needle the various probe point-to-point distribution on the surface of a substrate on the respective detection site. The fixed probe method has the following several.

(1) point-like fixed probe method: probe, through the aforesaid surface chemical is fixed on the gold or SiO₂ or other material on the detecting site. This kind of method is suitable for preparing low-density (per centimetre of about 100 the following probe) of the probe array.

(2) in-situ synthesizing probe method: directly on the corresponding detection site to synthesize the fixing the probe. In-situ synthesis of the probe is based on the principle of combined chemical synthesis. It through a set of positioning template to determine different chemical on the surface of substrate coupling site and order of monomers, such as by the four groups of 16 block template positioning synthesized to obtain 256 different four nucleotide probe array. 1st group in the shadow of the four template part respectively corresponding to the four different positions on the substrate, the template through different adenine, guanine, cytosine, chest pyrimidine (A, C, G, T) for four different base chemical coupling within the corresponding shadow regions. 2nd group of rolling with respect to the 1st group of rotating 90, the same method in the upper a layer of base are respectively coupled the four bases. At the end of the 2nd group of synthesis, on the substrate to prepare a total of 16 different two nuclear nucleotide. 3rd, four groups on the template to the template pattern, repeated 1st, method for synthesis of two groups, can produce 256 different four nucleotide probe array. As the template pattern subdivides the number of times the increase of n, can be generated on the chip 4²ⁿ kind of 2n base oligonucleotide probe. At present, many kinds of template technology has been used for in-situ synthesis of the probe DNA, such as optical to protect the parallel synthetic method, synthesis photoresist protection, microfluidic template dials the technique of the solid phase to, molecules such as the seal impression synthesis method a plurality of times. In-situ synthesis method can also be used for solid phase substrate on titanium or other macromolecule probe synthesis, can play the advantage of the micro-machining technique, very suitable for producing large-scale probe array chip, to realize high-density chip of standardization and mass production.

(3) the probe positioning synthetic method: this invention is through Figure 1-4 resistance element 2, without influencing the adjacent detection site of the set of the detection sites of the chip, the positioning of a probe synthesis. The selected resistance through the applied voltage, the detection site as the heat-activated in situ probe synthesis of widowed nucleotide chain. And, the synthesis reaction of the adjacent detecting pool of all resistance heating a large current, in order to make the non-synthetic the pool to keep the temperature above the temperature required in the synthesis, the synthesis reaction in the test pool. Similarly, the present invention automatically positioning sensor array can be set by the applied voltage of the electrode of the detection site, positioning in this detection pond electroluminescent or electrocatalytic synthesis reaction.

Moreover, the electrode set a detection site of the applied voltage can also be used to attract the pool of the reactant in the sample solution in order to accelerate the reaction speed. The target molecules, such as probe in the hybridization reaction can be added to the target specimen solution to the electrode voltage is applied to accelerate the reaction, the absence of an applied voltage, only target molecules of the sample solution through the diffusion reaches the probe, because of this kind of diffusion process efficiency is extremely low, usually require 1.5 to 2 hours to obtain the apparent reaction, but also most of the no response of the probe. Additional molecular charged target a voltage can be directly attracted to the connection of the probe on the electrode, so as to accelerate the reaction speed, and the number of the increased target/probe reaction. On the contrary, additional a reverse voltage, would elute unreacted and mismatched target molecule, this technology is suitable for all detection site built-in with an electrode of the chip. Furthermore, the applied voltage can be used to produce a shock current, is used for cleaning or passivation of the surface of the electrode.

Embodiment 2

The invention relates to an in-situ biochip, as shown in Figure 6A-6H shown, on the same by the substrate and an array of detection sites. See Figure 1, this embodiment can be kept or removing Figure 1 the RX1, RX2, RX3... RXM address lines, RY1, RY2, RY3... RYN address lines, X resistance logic circuit 3, Y resistance logic circuit 4.

The above-mentioned substrate 1, with the embodiment 1 are the same. On the substrate coated with a layer of SiO₂ film layer 5. In SiO₂ are M section along the X coordinate axis of the X address lines X1, X2, X3... address line and XM N along the Y axes of the Y address lines Y1, Y2, Y3... YN is staggered. The X address lines and Y address line beams MN with the X address lines and Y address lines of the detection sites is connected with, the above-mentioned M, N is a positive integer. Each detection site a plurality of the sample solution can contain 6 is in the shape of a circular hole formed by the concave of the pool 12. In all the pool 12 is covered with the polycrystalline silicon film on the wall of the hole 19, on the polycrystalline silicon film is coated with a metallization layer 20, the metallized layer is provided with electrode 8 and electrode 9, detection cell 12 of the bottom surface 18 and/or the electrodes are corrugated surface. The embodiment of the sensor, as with the embodiment of the structure of the 1 same.

The invention of the above-mentioned in-situ biochip preparation method is as follows.

1) preparation of the substrate. The material and method of the embodiment 1 the same.

2) on the substrate of the large scale integrated circuit technology to the manufacture of miniature sensor array.

(1) the substrate 1 is produced on thickness of about 5000 the of [...] SiO₂ film layer 5.

(2) the SiO₂ film layer 5 is, along the X coordinate axis is made of the X address lines and M section along the Y coordinate of the Y address lines N, legal leaves the hole hole pattern using photolithography again, the corrosion reaction ion etching method SiO₂ layer, each X address line and the Y address lines are MN the outside of a circular hole formed by the concave of the hole 17, the hole array a. All atractaspis hole depth of the hole of about 0.5 the  m, aperture of about 2 the  m, 2 the pitch between the holes  m.

(3) the use of chemical vapor-phase deposition method in SiO₂ film layer 5 is about the thickness of the 2000 [...] polycrystalline silicon film 19.

(4) reaction ion etching method is adopted, the hole 17 and on the upper surface of the bottom surface of the polysilicon film 19 part corrosion removing, a polycrystalline silicon film on the wall of the retaining hole.

(5) using W or Ti or Pt material, the silicidation reaction with the hole 17 of the hole wall of the polycrystalline silicon film is metallized, a metallization layer is made of 20,

(6) the use of chemical plating method, is characterized in that the metallization layer in the hole wall 20 is, Ni or Au material formed of the electrode 8 and electrode 9, the pool 12, a detection cell array. The can also be the bottom surface of the hole and/or the electrode surface, the corrugated surface is made.

(7) the pool of the electrode 8 and electrode 9 are respectively and each X address line and is connected with one end of the Y address, and the X address lines and Y address lines respectively connected with the other end of the common method for integrated in the substrate 1 is usually of the X addressing logic circuit 13 and Y addressing logic circuit 14 is connected, then the X addressing logic circuit and Y addressing logic circuit of the detection circuit 15 is connected, to form each sensor, composition sensor array.

(8) on the electrode of the sensor, fixing the probe 16. A detection site array.

3) is fixed on the detecting site method and embodiment of the probe 1 is the same.

The use of the invention, the in-situ bio-chip, such as the embodiment 1 and embodiment 2 of the target substance in-situ biological chip detection sensing detection has two major types: a type is based on the detecting site between the two electrodes of the dielectric loss, or AC admittance (impedance) or the change of the radio frequency loss of the transmission line; another kind is used for detecting the site based on the resonant frequency of the micro-resonator or quality factor (Q) changes. In the detection process of the invention, the in-situ bio-chip organic compound probe, biological molecule probe, cell probe, microbial probe in at least one combination of ceramics a display, can be captured at the same time, detection probe interaction with a target substance.

The use of the invention, the in-situ bio-chip electrical hybridization detection method: the invention, the in-situ bio-chip sensor array can be regarded as the gene for detecting each detection site 7 whether has the target gene. In the decoding DNA detection application, in each detection site a large number of shorter widowed nucleotide chain is fixed on the probe 16, one end of the probe chain surface with the detection site. To a sensor array, with each detection site probe chain different coding sequence, each detection site coding sequence of all probe chain is identical and known. When the chip of the present invention the detection of the detection site in the pool of unknown comprising the long-chain DNA (target) sample solution of 6 time, in the ideal situation, the target DNA will be only with its password sequence portion of the detection site complementary to a sequence on the probe closely widowed nucleotide chain , and not with any other detection site binding; in fact. Often the presence of certain weak DNA mismatch, but these mispairings can be through the appropriate ion concentration and the temperature of the sample solution using appropriate cleaning detecting pool be eliminated. Therefore, after cleaning many detection pool on the chip containing the combined into the hybridized DNA, in addition the remaining pool is still only contains the original widowed nucleotide chain probe. Each detection site of the through electrode 8 and 9 are sequentially telecommunications asked the pool, and record can be found which contains the hybrid DNA detection site. When the detecting site of hybridization on the DNA, its electrical properties will be significantly different. If the resonance frequencies of the DNA molecule. The former sample solution of the latter dielectric constant is about 10-100 times. The following detection method (2)-(5) that is each detection site based on the detected changes of the design. From this database, through the "iterative" or "neural network" algorithm reconstruct the intact coding sequence of that target DNA.

(1) loss factor method: Figure 7 is loss factor-frequency logarithmic graph, the graph of DNA (curve B) combined with the DNA binding (curve A) shows that the two loss factor (D) with the frequency (f) the change of the different, therefore, can be used to determine whether there is a detection site hybridization DNA. Loss factor detection can adopt very mature instrument method such as impedance meter , measuring instrument through X addressing logic circuit 13 and Y addressing logic circuit 14 sequentially with each detection site 7 is switched on.

(2) AC conductivity method : each detection site whether there is a hybrid DNA can also be detected by measuring the AC conductance, AC conductance G_AC = ε A/d, type in the dielectric constant of the electrode medium ε, A the effective area of the electrode, d is the effective distance between the electrodes. The sample solution to a DNA molecule, under in its relaxation frequency, the AC conductivity of the liquid without DNA is 100 times or more. Figure 9 is a schematic diagram of the principle of the method, in each detection site 7 of the electrode 8 and 9 is applied to a pulse or frequency scanning wave , in each a probe on an electrode 16, of the detection site picks measures the pond 12 canada has the target molecules in the sample solution. As shown in Figure 10, in a certain frequency is detected (DNA resonant frequency) lower G_AC great, indicating the existence of a hybrid DNA. Can be used in a certain frequency impedance meter lower measuring conductance G_L or resistance R_L (R_L = 1/G_L), the graph can also be such as 9, 10 of the note, in a certain frequency range the relationship between conductance and the frequency of the measurement.

(3) method for detecting the transmission-loss: in a transmission axis on the signal loss of the dielectric constant of the medium between the electrodes changes in ε sensitive. As shown in Figure 8 using wave address of the detection site plane shown in a schematic diagram, in each detection site between the Y address lines of the X address line and introduced into a transmission line 21, in each detection site 7, through the transmission line through the grading measuring a radio frequency loss of the electromagnetic wave, to complete electrical detection of hybrid molecules such as DNA. Transmission shaft axis can comprises a micro strip line, a microstrip, waveguide, a coplanar waveguide, a slot line or axis. In order to make this method has relatively high sensitivity, the pool 12 than Figure 4 the detecting the pond is wide or long, and the transmission line in the pool in the form of to the twists and turns with a maximum length.

(4) linear FM pulse detection method: as shown in Figure 11, in each of the detection sites is applied between two electrodes of a frequency scanning or voltage modulation waveform V_i, the resulting response waveform V₀ showing in Figure 12 or Figure 13, Figure in a frequency under (hybrid DNA frequency), the output voltage V₀ great, that the presence of a hybridization DNA. A frequency scanning waveform measuring hybridization DNA related to the relaxation frequency of the hybridized DNA can be obtained a total of the nature of certain information, such as cross-linked and non-cross-linked.

(5) method for detecting micro-mechanical resonator: in this, as shown in Figure 14, the substrate 1 is made of in a group of detection sites by the resonator. The resonator by the electrode film structure placed on the upper layer of the electrode 8 and disposed on the lower layer of the electrode 9 and the middle of the resonant cavity (medium is generally air) 22 form, electrode 9 material is metal, on the substrate 1 on the plane extending along the X direction, the electrode 8 is made of silicon nitride or tantalum, extending along the Y direction, the general size of electrode/long or wide as the diameter the 100  m. The electrode 8 is provided with a passage on the pool 12, the electrode in the tank 8 is fixed on the surface of the probe 16, the sample solution of target DNA 6 within the detection cell. Electrode 8 can be made of CVD technology to deposit a silicon nitride thin film, and can be controlled by the silicon-nitrogen ratio regulating room temperature and the evaporation temperature under the tension of the film, can be first in the substrate 1 is deposited on the silicon nitride thin film, of the substrate, a non-membrane 23, corrode, produce a window is formed the pool 12. The resonant frequency of resonator in thousands of Hz between several MHz, a very narrow resonance line width. When a radio frequency time of number when applied to the resonator. Will produce a high quality factor Q value, the response of the narrow line width. The resonator electrode 8 on the surface of the hybrid molecule Q value or the resonance frequency changes.

(6) method for detecting sound wave or electromagnetic wave: can also adopt the surface wave, such as a surface acoustic wave (SAW) or surface electromagnetic wave device which is similar to structure the resonant array detector. As shown in Figure 15, SAW resonant structure of the detector 24 by an acoustic transducer 25 and a SAW reflector 26 form, the transducer may be a lithium niobate crystal or quartz crystal substrate 27 the evaporation fork refersaluminum book membrane on the structure, the reflector can be made of an aluminum thin film grating, these graphical structure can adopt a standard lithography and deposition technology to process. The source 28 transmitting one frequency scanning wave W through acoustic medium base, the reflector the back reflected wave W ' through the pool 12, using instrument 29 the energy loss of the measuring transducer. Thereby detecting the resonant separate cavity induction by the reflector. Configuring the detection site on the medium 7, each detection site can have a corresponding transducer and reflector, or used in the substrate of a multi-position switch will all detection site and is connected with a transducer. Detection of target/probe sites will change its resonance frequency, can be detected by the detection site with the probe. Furthermore, through the detection site SAW wave phase of the transmission line can be used in the matrix compared with the reference transmission line, thereby measuring the surface phase changes due to the binding reaction, can also be used for determining the detection of a site binding molecule.